生存素反义寡核苷酸诱导结肠癌细胞株凋亡的实验研究

Experimental Study on Apoptosis Induced by Survivin-antisense Oligonucleotide in Colon Cancer Cell Lines

背景:生存素(survivin)是凋亡抑制蛋白家族的成员之一,在肿瘤组织和人类胚胎组织中有高水平表达,在分化成熟的组织中不表达或低水平表达。有研究显示,64.5%～85%的结肠癌组织中存在生存素基因表达,其表达与结肠癌患者的不良预后有关。目的:观察生存素反义寡核苷酸对结肠癌细胞株凋亡的影响。方法:通过基因工程技术构建生存素反义寡核苷酸质粒pcDNA3鄄sur鄄As,以脂质体转染的方法将质粒DNA转入结肠癌细胞株。应用逆转录聚合酶链反应(RT鄄PCR)和蛋白质印迹法(Westernblot)检测结肠癌细胞株中生存素mRNA和蛋白的表达,流式细胞仪和碘化丙啶(PI)染色检测细胞凋亡,比色测定检测caspase鄄3活性的变化。结果:SW1116、COLO205、HT鄄29和SW1417四株结肠癌细胞株中均有生存素mRNA和蛋白表达。瞬时转染生存素反义寡核苷酸后,4株结肠癌细胞株的细胞凋亡率均明显增加,并呈剂量依赖性(P<0.01);结肠癌细胞株SW1116在荧光显微镜下呈现典型的凋亡细胞形态,细胞中生存素蛋白表达水平明显降低,caspase鄄3活性显著增加(P<0.01)。结论:应用生存素反义寡核苷酸抑制生存素基因的表达能诱导结肠癌细胞株凋亡,以生存素为靶基因的治疗方案可能有助于结肠癌患者的治疗。

Survivin is a member of inhibitor of apoptosis protein family, which is highly expressed in cancerous tissues and during human embryonic and fetal development, but absent or very lowly expressed in all the mature tissues. Study had shown that survivin gene was expressed in 64.5%～85% of colonic cancerous tissues, and the expression of survivin was associated with unfavorable prognosis in colon cancer patients. Aims: To observe the effects of survivin-antisense oligonucleotide on the apoptosis of colon cancer cell lines. Methods: Through genetic engineering techniques, the survivin-antisense oligonucleotide plasmid pcDNA3-sur-As was constructed, then the plasmid DNA was transfected into colon cancer cell lines by Lipofectamine 2000. The survivin mRNA and protein expression levels in the colon cancer cell lines were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. Flow cytometry and propidium iodide (PI) staining were used to detect cell apoptosis. The variety of caspase-3 activity was determined by colorimetric assay. Results: Expression of survivin mRNA and protein were detected in the four tested colon cancer cell lines including SW1116, COLO 205, HT-29 and SW1417. After transient transfection of survivin-antisense oligo-nucleotide, the cell apoptosis rate was obviously increased in all colon cancer cell lines in a dose-dependent manner (P<0.01); Typical characteristics of apoptotic cells was shown in colon cancer cell line SW1116 under fluorescence microscope. The expression level of survivin protein in SW1116 cells was decreased, and the activity of caspase-3 was significantly increased (P<0.01). Conclusions: Inhibition of survivin gene expression by survivin-antisense oligonucleotide may induce apoptosis in colon cancer cell lines. Survivin targeted therapeutic protocol may possibly benefit the treatment of patients with colon cancer.